The present invention relates to a process for the differential spectroscopical and differential fluorometric determination of the interaction of substances with the aid of a microtitration plate and to the use of a microtitration plate for the differential spectroscopical and differential fluorometric determination of the interaction of substances.
The spectrophotometric determination of substances in homogeneous solutions is important in a large number of fields, e.g., in analyses of the environment, in basic research, in medical diagnostics, in forensic studies, etc. In the course of these determinations, substances such as nucleic acids, proteins, enzymes, their substrates, etc. are determined, ie., their presence is established and/or quantified. In these cases, it is often of interest to determine the interaction of various substances with each other. Differential spectrophotometry or differential fluorometry can be used to detect the interaction of substances with each other by way of their changed spectral or fluorometric properties. The spectral or fluorometric changes are caused either by direct interaction of the substances with each other or by binding induced configuration changes.
Similarly, solvent dependent configuration changes can be detected or aggregation equilibria can be observed in dependence upon the medium and the concentrations of substances.
Differential spectrophotometric or differential fluorometric measurements are usually made with the aid of two tandem cuvettes, namely a sample cell and a reference cell. A tandem cuvette comprises two chambers separated by a vertical partition. The solution of a first substance is put with identical concentrations into one chamber of each the sample cell and the reference cell for differential spectrophotometric or differential fluorometric measurements. The two other chambers are filled with buffer solution. In the subsequent measurement, which is made with a horizontal light beam passing through both chambers, identical spectrophotometric and fluorometric properties of the two tandem cuvettes must be observed. After that, a well defined amount of a second substance is added to the solution of the first substance in the sample cell and the same amount is filled into the buffer filled chamber of the reference cell in order to compensate for ligand absorption or ligand fluorescence. In order to avoid concentration differences, identical amounts of the buffer solution must be added to the solution of the first substance in the reference cell. After that, as described above, measurements are made a second time and the readings are compared. In view of the usually low spectral and fluorometric changes, this method is very sensitive in regard to concentration differences and therefore the respective additions of the second substance must be small as far as their volume is concerned. The limit of the determinations is given by the solubility of the substance in the buffer. Finally, when the substances are mixed, care must be taken that no solution is removed from the cells.
The technological problem underlying the present invention is to provide a process which overcomes the above cited shortcomings, in particular, minimises concentration effects in order to increase the sensitivity of the measurements, and which is capable of analysing substances of low solubility and of reducing errors due to mixing, as well as of facilitating rapid, universally applicable, parallel and therefore low cost determinations of interactions of substances.
The present invention solves the technological problem by providing a process for determining the differential spectroscopical and fluorometric interaction of at least two different substances by means of a microtitration plate containing a plurality of small bowls, wherein a lid with at least one trough associated with one small bowl is associated with the microtitration plate, and wherein a first solution of a first substance is placed into a small bowl, a second solution of a second substance is placed into the trough associated with the small bowl, and a third solution containing a mixture of the first substance and the second substance is placed into a further trough and/or further small bowl so that subsequently the differential spectroscopical and/or differential fluorometric determination can be carried out by vertical irradiation through the filled trough and the small bowls with electromagnetic waves. Conventional photometers and fluorometers, ie., ELISA or MTP readers can be used for these measurements. Thus, the invention provides that two different substances are arranged separated from each other in vertically superimposed relationship in a region characterised as the reference region, whereas the mixture of the substances is situated in a sample region of the microtitration plate and can be analysed at the same time.
If the first substance interacts with the second substance, accompanied by a change in the spectral and/or fluorometric properties, the extinction coefficients or the fluorescence characteristics of at least one of the substances in the third solution changes, ie., in the mixture of the two solutions, in comparison with the reading obtained from the two separate solutions. These changes in the extinction coefficient allow conclusions about the substance interactions and the substance properties and structures. The extinction coefficients or the fluorescence characteristics are constant if there is no interaction or in null balance.
With the identical geometrical features preferred in accordance with the invention for the small bowls of the microtitration plate and the troughs in the lid, there are identical absorptions in the sample region, ie., in the region of the microtitration plate in which the trough containing the mixture of the first and the second solution is situated, and the reference region, ie., in the region of the microtitration plate in which the first and the second solution are arranged separated from each other and in superimposed relationship in trough and small bowls, provided that no solutions were filled in. If the geometrical features.of the respective small bowls or troughs are nonidentical, a geometrical factor must be introduced in the determination of the absorption.
In a particularly preferred embodiment, the invention relates to the aforementioned process, wherein the third solution is provided by mixing a solution of the first substance with a solution of the second substance, with the volume and/or substance concentrations of the two solutions used for the preparation of the third solution each being equal to those of the first and of the second solution.
In a further preferred embodiment, the invention relates to an aforementioned process, wherein the height of the third solution layer in the small bowl or the trough containing the third solution is equal to the sum of the heights of the layers of the first and the second solution in the small bowl and in the trough in which these solutions are situated.
In a further preferred embodiment. the invention provides that the heights of the filled in first, second, and/or third solutions are standardised, ie., that the development of menisci or reading errors resulting from different levels of filling of the solutions are precluded by using in accordance with the invention a lid with at least one trough, wherein the base section of this trough ensures a well defined length of the light path in the small bowl from the base portion of the small bowl to the base section of the trough arranged on top.
The present invention envisages the use of a microtitration plate containing a plurality of small bowls, with at least part of the microtitration plate covered by a lid having at least one trough associated with a small bowl. The essential features of such sample carrier systems have been described, for example, in DE 44 05 375 A1 which, as far as the production and the structure of such sample carrier systems is concerned, is included in the present disclosure.
Thus, the invention makes use of a sample carrier system comprising two elements, namely a microtitration plate and a lid associated with this microtitration plate.
The lid includes at least one trough having one or more sidewalls and a base section, preferably a plurality of troughs, each formed by sidewalls and a base section and interconnected with the others via a base plate and/or frame member. The lid can cover the microtitration plate partially or completely, and it can be provided that only regions of the lid have troughs with which small bowls are associated, with other regions of the lid being planar. The lid is associated with the microtitration plate in a fashion such that it can be plugged onto the same or laid down on the same, with the troughs of the lid extending from above into the openings of the small bowls of the microtitration plate or engaging the same, ie., being associated with the same. However, the lid can also be adapted to be folded up or folded down at the microtitration plate, e.g., by means of hinges, can be integral with the microtitration plate or can be associated permanently with the same in some other way, wherein one opening for filling the small bowls must be provided in each of the latter two embodiments.
It can be provided in one embodiment that the lid has a single trough composed of a base section, sidewalls, and, optionally, a circumferential folded rim or collar, wherein the base plate of the lid covers in planar fashion the remaining part of the microtitration plate either completely or partially. This lid with a single trough can be used in combination with various microtitration plates. When microtitration plates with the conventional shape of the small bowls or appropriately shaped small bowls of novel microtitration plates. are used, the upper rim or the collar of the lid serves as a support surface on the microtitration plate. Also the bottom of the lid of the present invention can be used as a support surface on the microtitration plate with an appropriately adapted shape of the small bowls of the microtitration plate.
The base plate of the lid can be configured as a web and/or advantageously comprises a frame member fully or partially embracing the microtitration plate, with the frame member serving for plugging the lid onto a microtitration plate and keeping the lid in stationary position on the microtitration plate. The lid can bear troughs arranged in the form of a row, ie., it can be composed of troughs arranged in succession. However, it can also be provided that the lid comprises troughs in the form of a matrix. Of course, the invention also provides that such a lid covers only regions, e.g., a row on a microtitration plate, le., that it is associated with the same. Accordingly, a lid, which makes up a row, can be associated with a row of small bowls on a microtitration plate. But it can also be provided to associate a lid configured as a matrix with a microtitration plate in the form of a matrix, with the lid covering all the small bowls of the microtitration plate. The specific form of the lid depends upon the form of the conventional or novel microtitration plate used.
It is particularly preferred to practice the process with a microtitration lid which covers some of the small bowls of the microtitration plate in planar fashion, ie., without troughs, and forms in his way a sample region of the subjacent microtitration plate, with an other part of the lid having troughs associated with individual small bowls and covering the reference region of the microtitration plate. In the sample region of the microtitration plate, where therefore no superimposed positioning of samples is allowed, the third solution, ie., the mixture of the first and the second substance, is filled in, whereas the first and the second solution can be filled in superimposed relationship and separated from each other into the small bowls of the reference region into which the troughs extend.
Of course, it is also possible to provide a lid which has troughs in the sample region of the microtitration plate. In this case, it can also be provided in accordance with the invention to fill the third solution only into the trough of the lid rather than into the small bowl proper or into both, ie., into the small bowl and the associated trough.
It is a feature of the lids used in accordance with the invention that they provide one trough for each of the small bowls associated with the trough so that the base section of the trough is arranged in, or on, the opening of the small bowl and provides a well defined clear gap or distance between the bottom portion of the small bowl and the bottom portion of the trough, which gap is partially or completely filled with the sample liquid. Accordingly, there is advantageously provided a well defined layer thickness or layer height of the sample to be analysed because the light beam passes vertically over a well defined path within the sample, without being influenced by the level of filling and the development of menisci. The trough therefore must be configured so that no sample liquid can enter from the small bowl into the trough, ie., the trough must be downwardly and laterally enclosed in liquid impermeable fashion and, accordingly, must be enclosed, for example, by integral sidewalls and a base section, with an upward opening advantageously remaining available. The size and the geometrical form of the troughs are arbitrary and depend particularly upon the size and the geometrical form of the small bowls. According to the invention, it can also be provided to configure the trough of the lid, for example, as a hollow body which is enclosed on all sides and has an opening for filling.
The clear height of the small bowls of the microtitration plates, ie., the distance from the bottom of the small bowl to its upper edge, is reduced by the troughxe2x80x3base section which enters into the small bowl. Since it can be provided in advantageous fashion that all, or many, troughs of the lid have the same dimensions and geometrical forms as the small bowls, all the small bowls, which are partially covered by the base sections of the troughs, are standardised in regard to the path length of the light beam passing vertically through the small bowls, ie., the layer thickness or the layer height, ie., the distance travelled by the vertically passing light beam within the completely filled small bowls or the troughs is given the same value in all covered small bowls, whereby possible differences in the level of filling or the development of menisci are compensated. It can be provided in particularly advantageous fashion that the cross section area of the small bowl, ie., the area perpendicular to the height of filling, is on the level of the small bowl (on which the base section of the trough is situated) greater than the area of the base section of the trough. As a consequence, the base section does not cover the entire clear width of the small bowl in its opening but only part thereof so that sample liquid present in the small bowls can be expelled upwards, whereby nevertheless a light path standardised in regard to its length is ensured.
In the context of the present invention, a microtitration plate is defined as a plate containing a plurality of small bowls, with the small bowls serving to accommodate samples which are subjected to spectrophotometric or fluorometric measurements. The plate and small bowls can be integral with each other but it is also possible to provide that the small bowls are arranged in removable or non removable fashion as separate units or as combined units in a plate and/or frame member. Accordingly, plates and or frames are configured as support means for cuvettes or small bowls or cuvette units or cuvette combinations. The small bowls can be configured as simple depressions, wells, troughs, cuvettes, tubes or the like, and also their cross section shape is arbitrary and therefore can be, for example, circular, polygonal, square, rectangular or oval. The number of small bowls in each microtitration plate is arbitrary, and the small bowls are usually arranged in the form of a matrix, comprising, for example, 96, 384 or 1536 small bowls. In connection with the present invention, a microtitration plate is also understood as an arrangement of small bowls in a row, ie., not in the form of a matrix, and, accordingly, plates having, for example, 8 small bowls or plates having 8 small bowls and being interconnected by web sections are also defined as microtitration plates. The small bowls of the microtitration plate in general have an upward opening so that the samples can be poured in. The small bowls are delimited laterally by sidewalls and downwards by a bottom portion; these elements can be integral with each other.
In connection with the present invention, a substance is understood as a material to be determined on which measurements can be made by spectrophotometric or fluorometric processes, ie., which can be detected and/or quantified, ie., which acts as a chromophore. Such a material to be determined can be formed, for example, by a protein, a peptide, a protein or peptide derivative, a metal complex, a ligand, a dyestuff, a nucleic acid or some other low molecular or macromolecular compound. The microtitration plates to be used in accordance with the invention can be made from any material, e.g., from polyvinylchloride, polystyrene, polypropylene, quartz glass or the like. Of course, it can also be provided to use microtitration plates with coated small bowls.
Thus, the present invention provides a process which facilitates universally applicable, rapid, parallel and therefore low cost studies of interactions of various substances. It is therefore advantageously feasible to determine properties of substances having short half lives. A further advantage results from the fact that concentration effects, which reduce the sensitivity, are minimised and that, furthermore, substances of low solubility can be studied in a better way. Finally, errors due to mixing, which occur in conventional processes, are reduced.
The invention relates also to one of the above described microtitration plates, ie., to a microtitration plate having a plurality of small bowls, with the microtitration plate being associated with a lid having at least one trough associated with a small bowl, with the lid comprising a lid base plate having at least one trough and a preferably substantially flat upper lid plate, and with the upper lid plate adapted to be moved vertically relative to the lid base plate and serving, for covering the trough in the lid base plate. Such a device is advantageous insofar as well defined layer thicknesses can be obtained in both the small bowls of the microtitration plate and the trough of the lid.
The invention also relates to the use of a microtitration plate bearing a plurality of small bowls and a lid, with the lid having at least one trough associated with a small bowl for practising a process for the differential spectroscopical or differential fluorometric measurement of the interaction of substances, with the lid being composed preferably of two plates adapted to be moved vertically relative to each other and with the lower plate containing the troughs.